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Research & Scholarship

Current Research and Scholarly Interests

Our Neuromuscular Division organizes a comprehensive effort to combat and conquer diseases of the peripheral nerves and muscles, including the muscular dystrophies (myotonic, Duchenne, limb girdle, facioscapulohumeral, and congenital muscular dystrophies), motor neuron disorders (ALS and SMA), neuromuscular junction disease (MG, CMS), and peripheral neuropathies (CMT, CIDP). While keeping the patients and families foremost in mind, our research seeks to: define and understand genetic causes; clarify the molecular and cellular consequences of genetic change; determine the multisystemic features that are underappreciated but clinically significant consequence of these diseases; develop and improve methods for managing and treating each disease.

We have identified the genetic cause of several neuromuscular disorders, most notably myotonic dystrophy type 2, which we continue to study to advance understanding of all forms of myotonic dystrophy. We have also contributed to genetic understanding of Duchenne muscular dystrophy, and other muscle and ataxic disorders. We are continuing to investigate the epigenetic and molecular consequences of these diseases through investigation of patient-derived specimens.

We have focused on defining the central nervous system features of neuromuscular disorders, which severely impact patients and families but have been incompletely investigated, explained or managed. Detailed neuropsychological and brain MRI studies are helping to define the developmental and progressive CNS aspects of these conditions, for which we then seek molecular and cellular explanations through cell-based studies of patient-derived specimens.

To assure our research is translatable to clinical practice, we are simultaneously involved in collaborative clinical research on novel treatments for neuromuscular disease, including antisense oligonucleotides and pharmacologic manipulation of muscle function, viral gene therapies and cell-based treatments.

In summary, we work with patients to define neuromuscular disorders more rigorously and understand them more thoroughly, so novel treatments will successfully combat these devastating disorders.

Clinical Trials

Study to focus on the defining and managing the neuropsychological abnormalities of myotonic
dystrophy and to find out if the neuropsychological abnormalities have any correlation with
changes seen on Magnetic Resonance Imaging.

A Study to Assess the Efficacy, Safety and Pharmacokinetics of Nusinersen (ISIS 396443) in Infants With Spinal Muscular Atrophy (SMA)Not Recruiting

The primary objective is to examine the clinical efficacy of multiple doses of nusinersen
(ISIS 396443) administered intrathecally to participants with Infantile-Onset Spinal Muscular
Atrophy (SMA). The secondary objectives are to examine the safety and tolerability of
multiple doses of nusinersen administered intrathecally to participants with infantile-onset
SMA and to examine the cerebral spinal fluid (CSF) and plasma Pharmacokinetics (PK) of
multiple doses of nusinersen administered intrathecally to participants with infantile-onset
SMA.

Stanford is currently not accepting patients for this trial.For more information, please contact Shirley Paulose, MBBS, MS, 650-725-4341.

This project includes two projects. One is looking for new genes that cause Charcot Marie
Tooth disease (CMT). The other is looking for genes that do not cause CMT, but may modify the
symptoms a person has.

The purpose of the study is to determine the best ways to assess how people are affected by
myotonic dystrophy type 1 (DM1). The study will assess walking speed, muscle strength, muscle
size, myotonia, heart rhythm, mental efficiency, and overall health. Participants will
complete questionnaires to record their ideas about how they are affected by DM1. The study
will evaluate people with DM1 over 1 year to determine how the condition changes over time.
The study will identify biomarkers of DM1. Biomarkers are laboratory measurements that show
the effects of DM1 on a person's muscle tissue or blood. Biomarkers are needed in future
studies to determine how DM1 may respond to treatments.

The main objective of this study is to evaluate the efficacy of SRP-4045 and SRP-4053
compared to placebo in Duchenne muscular dystrophy (DMD) patients with out-of-frame deletion
mutations amenable to skipping exon 45 and exon 53, respectively.

This is an observational longitudinal study to determine the natural history and
genotype-phenotype correlations of disease causing mutations in Charcot Marie Tooth disease
(CMT) type 1B (CMT1B), 2A (CMT2A), 4A (CMT4A), and 4C (CMT4C).
The investigators will also be determine the capability of the newly developed CMT Pediatric
Scale (CMT Peds scale) and the Minimal Dataset to measure impairment and perform longitudinal
measurements in patients with multiple forms of CMT over a five year window

Stanford is currently not accepting patients for this trial.For more information, please contact Carly E Siskind, MS, 650-721-5588.

Spinal Muscular Atrophy (SMA) is the leading genetic cause of death in infancy. It is a
devastating disease that leads to progressive loss of those nerve cells that control our
muscle bulk and movement. Patients develop increasing weakness in all muscles, eventually
including those needed for breathing. In more than half of patients, SMA starts in infancy
and typically leads to death within the first 2 years of life. In others, the disease begins
in childhood and leads to significant disability.
SMA is caused by a defect in the "Survival of Motor Neurons" (SMN1) gene. Researchers are
hopeful to find a cure, because nature has provided humans with a second gene, almost an
identical copy of the SMN1 gene. Normally, the second gene does not contribute much, but
researchers think that its function can be increased by medications.
To find out whether these medications help patients with SMA, we have to conduct clinical
trials. Here, we propose to prepare for clinical trials. We will invite SMA patients to join
our research effort. We will examine them regularly to better understand their disease. The
visits will include questions, physical exam, blood drawing, and sometimes X-rays and a skin
biopsy. We will use modern computer methods to process the information. While we are doing
this, we will plan a clinical trial. Once the clinical trial begins, we will offer SMA
patients participation if they meet the criteria for that trial.
We will make sure that the participants' privacy is maintained and that the study risks are
as low as possible.
Identifying an effective SMA treatment is very important because there is currently none.
Clinical trials are the only way to decide whether a new treatment works in SMA patients or
not.

Stanford is currently not accepting patients for this trial.For more information, please contact Spectrum Child Health, 650-724-1175.

The primary objective is to evaluate the long-term safety and tolerability of nusinersen
(ISIS 396443) administered by intrathecal (IT) injection to participants with Spinal Muscular
Atrophy (SMA) who previously participated in investigational studies of nusinersen. The
secondary objective is to examine the long-term efficacy of nusinersen administered by IT
injection to participants with SMA who previously participated in investigational studies of
nusinersen.

The "Clinical Outcome Study for Dysferlinopathy" is being performed in centres in Europe (UK-
Newcastle; Spain- Barcelona, Sevilla; Germany- Berlin, Munich; Italy- Padova; France- Paris,
Marseille), USA (Charlotte, NC; Columbus, OH; Washington, DC; St.Louis, MO, Stanford CA),
Japan (Tokyo) and Australia (Sydney). Oversight and funding for this study is being provided
by the Jain Foundation, a non-profit foundation dedicated to finding therapies for
dysferlinopathies(LGMD2b/Miyoshi). The aim of this "Clinical Outcome Study" is to determine
the clinical outcome measures required for future clinical trials, characterize the disease
progression of dysferlinopathy and collect biological samples for the identification of
disease markers that are needed to non-invasively monitor the disease during clinical trials.
Without this information, effective clinical trials cannot be performed.
This study is recruiting a large number of genetically confirmed dysferlinopathy patients
aged 10 years or older, who are ambulant or non-ambulant. Participants will be assessed at 6
visits over 3 years via medical, physiotherapy, and MRI/MRS assessments, as well as standard
blood tests. Optionally, the participants can donate blood samples and a skin sample for use
in the identification of disease markers and other approved research.

Stanford is currently not accepting patients for this trial.For more information, please contact Spectrum Child Health, 650-724-1175.

Abstract

Reports on the clinical meaningfulness of outcome measures in spinal muscular atrophy (SMA) are rare. In this two-part study, our aim was to explore patients' and caregivers' views on the clinical relevance of the Hammersmith Functional Motor Scale Expanded- (HFMSE).First, we used focus groups including SMA patients and caregivers to explore their views on the clinical relevance of the individual activities included in the HFMSE. Then we asked caregivers to comment on the clinical relevance of possible changes of HFMSE scores over time. As functional data of individual patients were available, some of the questions were tailored according to their functional level on the HFMSE.Part 1: Sixty-three individuals participated in the focus groups. This included 30 caregivers, 25 patients and 8 professionals who facilitated the discussion. The caregivers provided a comparison to activities of daily living for each of the HFMSE items. Part 2: One hundred and forty-nine caregivers agreed to complete the questionnaire: in response to a general question, 72% of the caregivers would consider taking part in a clinical trial if the treatment was expected to slow down deterioration, 88% if it would stop deterioration and 97% if the treatment was expected to produce an improvement. Caregivers were informed of the first three items that their child could not achieve on the HFMSE. In response 75% indicated a willingness to take part in a clinical trial if they could achieve at least one of these abilities, 89% if they could achieve two, and 100% if they could achieve more than 2.Our findings support the use of the HFMSE as a key outcome measure in SMA clinical trials because the individual items and the detected changes have clear content validity and clinical meaningfulness for patients and their caregivers.

Abstract

Recent translational research developments in Spinal Muscular Atrophy (SMA), outcome measure design and demands from regulatory authorities require that clinical outcome assessments are 'fit for purpose'. An international collaboration (SMA REACH UK, Italian SMA Network and PNCRN USA) undertook an iterative process to address discontinuity in the recorded performance of the Hammersmith Functional Motor Scale Expanded and developed a revised functional scale using Rasch analysis, traditional psychometric techniques and the application of clinical sensibility via expert panels. Specifically, we intended to develop a psychometrically and clinically robust functional clinician rated outcome measure to assess physical abilities in weak SMA type 2 through to strong ambulant SMA type 3 patients. The final scale, the Revised Hammersmith Scale (RHS) for SMA, consisting of 36 items and two timed tests, was piloted in 138 patients with type 2 and 3 SMA in an observational cross-sectional multi-centre study across the three national networks. Rasch analysis demonstrated very good fit of all 36 items to the construct of motor performance, good reliability with a high Person Separation Index PSI 0.98, logical and hierarchical scoring in 27/36 items and excellent targeting with minimal ceiling. The RHS differentiated between clinically different groups: SMA type, World Health Organisation (WHO) categories, ambulatory status, and SMA type combined with ambulatory status (all p < 0.001). Construct and concurrent validity was also confirmed with a strong significant positive correlation with the WHO motor milestones rs = 0.860, p < 0.001. We conclude that the RHS is a psychometrically sound and versatile clinical outcome assessment to test the broad range of physical abilities of patients with type 2 and 3 SMA. Further longitudinal testing of the scale with regards change in scores over 6 and 12 months are required prior to its adoption in clinical trials.

Abstract

Nusinersen is a 2'-O-methoxyethyl phosphorothioate-modified antisense drug being developed to treat spinal muscular atrophy. Nusinersen is specifically designed to alter splicing of SMN2 pre-mRNA and thus increase the amount of functional survival motor neuron (SMN) protein that is deficient in patients with spinal muscular atrophy.This open-label, phase 2, escalating dose clinical study assessed the safety and tolerability, pharmacokinetics, and clinical efficacy of multiple intrathecal doses of nusinersen (6 mg and 12 mg dose equivalents) in patients with infantile-onset spinal muscular atrophy. Eligible participants were of either gender aged between 3 weeks and 7 months old with onset of spinal muscular atrophy symptoms between 3 weeks and 6 months, who had SMN1 homozygous gene deletion or mutation. Safety assessments included adverse events, physical and neurological examinations, vital signs, clinical laboratory tests, cerebrospinal fluid laboratory tests, and electrocardiographs. Clinical efficacy assessments included event free survival, and change from baseline of two assessments of motor function: the motor milestones portion of the Hammersmith Infant Neurological Exam-Part 2 (HINE-2) and the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) motor function test, and compound motor action potentials. Autopsy tissue was analysed for target engagement, drug concentrations, and pharmacological activity. HINE-2, CHOP-INTEND, and compound motor action potential were compared between baseline and last visit using the Wilcoxon signed-rank test. Age at death or permanent ventilation was compared with natural history using the log-rank test. The study is registered at ClinicalTrials.gov, number NCT01839656.20 participants were enrolled between May 3, 2013, and July 9, 2014, and assessed through to an interim analysis done on Jan 26, 2016. All participants experienced adverse events, with 77 serious adverse events reported in 16 participants, all considered by study investigators not related or unlikely related to the study drug. In the 12 mg dose group, incremental achievements of motor milestones (p<0·0001), improvements in CHOP-INTEND motor function scores (p=0·0013), and increased compound muscle action potential amplitude of the ulnar nerve (p=0·0103) and peroneal nerve (p<0·0001), compared with baseline, were observed. Median age at death or permanent ventilation was not reached and the Kaplan-Meier survival curve diverged from a published natural history case series (p=0·0014). Analysis of autopsy tissue from patients exposed to nusinersen showed drug uptake into motor neurons throughout the spinal cord and neurons and other cell types in the brainstem and other brain regions, exposure at therapeutic concentrations, and increased SMN2 mRNA exon 7 inclusion and SMN protein concentrations in the spinal cord.Administration of multiple intrathecal doses of nusinersen showed acceptable safety and tolerability, pharmacology consistent with its intended mechanism of action, and encouraging clinical efficacy. Results informed the design of an ongoing, sham-controlled, phase 3 clinical study of nusinersen in infantile-onset spinal muscular atrophy.Ionis Pharmaceuticals, Inc and Biogen.

Abstract

There is a growing need for a robust clinical measure to assess upper limb motor function in spinal muscular atrophy (SMA), as the available scales lack sensitivity at the extremes of the clinical spectrum. We report the development of the Revised Upper Limb Module (RULM), an assessment specifically designed for upper limb function in SMA patients.An international panel with specific neuromuscular expertise performed a thorough review of scales currently available to assess upper limb function in SMA. This review facilitated a revision of the existing upper limb function scales to make a more robust clinical scale.Multiple revisions of the scale included statistical analysis and captured clinically relevant changes to fulfill requirements by regulators and advocacy groups.The resulting RULM scale shows good reliability and validity, making it a suitable tool to assess upper extremity function in the SMA population for multi-center clinical research. This article is protected by copyright. All rights reserved.

Abstract

Outcomes sensitive to change over time in non-ambulatory boys/men with Duchenne muscular dystrophy (DMD) are not well-established.Subjects (n = 91; 16.8 ± 4.5 years old) were assessed at baseline and 6-month intervals for 2 years. We analyzed all subjects using an intent-to-treat model and a subset of stronger subjects with Brooke Scale score ≤4, using repeated measures.Eight patients (12-33 years old) died during the study. Sixty-six completed 12-month follow-up, and 51 completed 24-month follow-up. Those taking corticosteroids performed better at baseline, but rates of decline were similar. Forced vital capacity percent predicted (FVC% predicted) declined significantly only after 2 years. However, Brooke and Egen Klassifikation (EK) Scale scores, elbow flexion, and grip strength declined significantly over both 1 and 2 years.Brooke and EK Scale scores, elbow flexion, and grip strength were outcomes most responsive to change. FVC% predicted was responsive to change over 2 years. Corticosteroids benefited non-ambulatory DMD subjects but did not affect decline rates of measures tested here. Muscle Nerve 54: 681-689, 2016.

Abstract

New developments in the rapid diagnosis and treatment of boys with Duchenne muscular dystrophy (DMD) have led to growing enthusiasm for instituting DMD newborn screening (NBS) in the United States. Our group has been interested in developing clinical guidance to be implemented consistently in specialty care clinics charged with the care of presymptomatically identified newborns referred after DMD-NBS. We reviewed the existing literature covering patient-centered clinical follow-up after NBS, educational material from public health and advocacy sites, and federal recommendations on effective NBS follow-up. We discussed the review as a group and added our own experience to develop materials suitable for initial parent and primary care provider education. These materials and a series of templates for subspecialist encounters could be used to provide consistent care across centers and serve as the basis for ongoing quality improvement. Muscle Nerve 54: 186-191, 2016.

Abstract

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

Abstract

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Abstract

To characterize 2 novel TRPV4 mutations in 2 unrelated families exhibiting the Charcot-Marie-Tooth disease type 2C (CMT2C) phenotype.Direct CMT gene testing was performed on 2 unrelated families with CMT2C. A 4-fold symmetric tetramer model of human TRPV4 was generated to map the locations of novel TRPV4 mutations in these families relative to previously identified disease-causing mutations (neuropathy, skeletal dysplasia, and osteoarthropathy). Effects of the mutations on TRPV4 expression, localization, and channel activity were determined by immunocytochemical, immunoblotting, Ca(2+) imaging, and cytotoxicity assays.Previous studies suggest that neuropathy-causing mutations occur primarily at arginine residues on the convex face of the TRPV4 ankyrin repeat domain (ARD). Further highlighting the key role of this domain in TRPV4-mediated hereditary neuropathy, we report 2 novel heterozygous missense mutations in the TRPV4-ARD convex face (p.Arg237Gly and p.Arg237Leu). Generation of a model of the TRPV4 homotetramer revealed that while ARD residues mutated in neuropathy (including Arg237) are likely accessible for intermolecular interactions, skeletal dysplasia-causing TRPV4 mutations occur at sites suggesting disruption of intramolecular and/or intersubunit interactions. Like previously described neuropathy-causing mutations, the p.Arg237Gly and p.Arg237Leu substitutions do not alter TRPV4 subcellular localization in transfected cells but cause elevations of cytosolic Ca(2+) levels and marked cytotoxicity.These findings expand the number of ARD residues mutated in TRPV4-mediated neuropathy, providing further evidence of the central importance of this domain to TRPV4 function in peripheral nerve.

Abstract

We aimed to characterize genotype-phenotype correlations and establish baseline clinical data for peripheral neuropathies caused by mutations in the myelin protein zero (MPZ) gene. MPZ mutations are the second leading cause of Charcot-Marie-Tooth disease type 1. Recent research makes clinical trials for patients with MPZ mutations a realistic possibility. However, the clinical severity varies with different mutations and natural history data on progression is sparse. We present cross-sectional data to begin to define the phenotypic spectrum and clinical baseline of patients with these mutations. A cohort of patients with MPZ gene mutations was identified in 13 centres of the Inherited Neuropathies Consortium - Rare Disease Clinical Research Consortium (INC-RDCRC) between 2009 and 2012 and at Wayne State University between 1996 and 2009. Patient phenotypes were quantified by the Charcot-Marie-Tooth disease neuropathy score version 1 or 2 and the Charcot-Marie-Tooth disease paediatric scale outcome instruments. Genetic testing was performed in all patients and/or in first- or second-degree relatives to document mutation in MPZ gene indicating diagnosis of Charcot-Marie-Tooth disease type 1B. There were 103 patients from 71 families with 47 different MPZ mutations with a mean age of 40 years (range 3-84 years). Patients and mutations were separated into infantile, childhood and adult-onset groups. The infantile onset group had higher Charcot-Marie-Tooth disease neuropathy score version 1 or 2 and slower nerve conductions than the other groups, and severity increased with age. Twenty-three patients had no family history of Charcot-Marie-Tooth disease. Sixty-one patients wore foot/ankle orthoses, 19 required walking assistance or support, and 10 required wheelchairs. There was hearing loss in 21 and scoliosis in 17. Forty-two patients did not begin walking until after 15 months of age. Half of the infantile onset patients then required ambulation aids or wheelchairs for ambulation. Our results demonstrate that virtually all MPZ mutations are associated with specific phenotypes. Early onset (infantile and childhood) phenotypes likely represent developmentally impaired myelination, whereas the adult-onset phenotype reflects axonal degeneration without antecedent demyelination. Data from this cohort of patients will provide the baseline data necessary for clinical trials of patients with Charcot-Marie-Tooth disease caused by MPZ gene mutations.

Abstract

The international Inherited Neuropathy Consortium (INC) was created with the goal of obtaining much needed natural history data for patients with Charcot-Marie-Tooth (CMT) disease. We analysed clinical and genetic data from patients in the INC to determine the distribution of CMT subtypes and the clinical impairment associated with them.We analysed data from 1652 patients evaluated at 13 INC centres. The distribution of CMT subtypes and pathogenic genetic mutations were determined. The disease burden of all the mutations was assessed by the CMT Neuropathy Score (CMTNS) and CMT Examination Score (CMTES).997 of the 1652 patients (60.4%) received a genetic diagnosis. The most common CMT subtypes were CMT1A/PMP22 duplication, CMT1X/GJB1 mutation, CMT2A/MFN2 mutation, CMT1B/MPZ mutation, and hereditary neuropathy with liability to pressure palsy/PMP22 deletion. These five subtypes of CMT accounted for 89.2% of all genetically confirmed mutations. Mean CMTNS for some but not all subtypes were similar to those previously reported.Our findings confirm that large numbers of patients with a representative variety of CMT subtypes have been enrolled and that the frequency of achieving a molecular diagnosis and distribution of the CMT subtypes reflects those previously reported. Measures of severity are similar, though not identical, to results from smaller series. This study confirms that it is possible to assess patients in a uniform way between international centres, which is critical for the planned natural history study and future clinical trials. These data will provide a representative baseline for longitudinal studies of CMT.ID number NCT01193075.

Abstract

Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients.Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions.As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype.The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone.

Abstract

The alpha-dystroglycanopathies are genetically heterogeneous muscular dystrophies that result from hypoglycosylation of alpha-dystroglycan (α-DG). Alpha-dystroglycan is an essential link between the extracellular matrix and the muscle fiber sarcolemma, and proper glycosylation is critical for its ability to bind to ligands in the extracellular matrix. We sought to identify the genetic basis of alpha-dystroglycanopathy in a family wherein the affected individuals presented with congenital muscular dystrophy, brain abnormalities and generalized epilepsy. We performed whole exome sequencing and identified compound heterozygous GMPPB mutations in the affected children. GMPPB is an enzyme in the glycosylation pathway, and GMPPB mutations were recently linked to eight cases of alpha-dystroglycanopathy with a range of symptoms. We identified a novel mutation in GMPPB (p.I219T) as well as a previously published mutation (p.R287Q). Thus, our work further confirms a role for GMPPB defects in alpha-dystroglycanopathy, and suggests that glycosylation may play a role in the neuronal membrane channels or networks involved in the physiology of generalized epilepsy syndromes. This article is part of a Special Issue entitled RNA Metabolism 2013.

Abstract

Cerebral involvement in Myotonic Dystrophy Type 1 (DM1) is well-established but not well characterized. This study applied new Diffusion Tensor Imaging (DTI) tractography to characterize white matter disturbance in adults with DM1. Forty-five participants with DM1 and 44 control participants had MRIs on a Siemens 3T TIM Trio scanner. Data were processed with TRActs Constrained by UnderLying Anatomy (TRACULA) and 7 tracts were evaluated. Bilateral disturbances in white matter integrity were seen in all tracts in participants with DM1 compared to controls. There were no right-left hemisphere differences. The resulting DTI metrics were correlated with cognitive functioning, particularly working memory and processing speed. Motor speed was not significantly correlated with white matter microstructural integrity and, thus, was not the core explanation for the working memory and processing speed findings. White matter integrity was correlated with important clinical variables including the muscular impairment rating scale (MIRS). CTG repeat length was moderately associated with white matter status in corticospinal tract and cingulum. Sleepiness (Epworth Sleepiness Scale) was moderately associated with white matter status in the superior longitudinal fasciculus and cingulum. Overall, the results add to an emerging literature showing widespread white matter disturbances in both early-onset and adult-onset DM1. Results suggest that further investigation of white matter pathology is warranted in DM1 and that non-invasive measures such as DTI have a potentially important clinical value in characterizing the status of individuals with DM1.

Abstract

The pathogenesis of Duchenne muscular dystrophy starts before birth. Despite this, clinical trials exclude young boys because traditional outcome measures rely on cooperation. We recently used the Bayley-III Scales of Infant and Toddler Development to study 24 infants and boys with Duchenne muscular dystrophy. Clinical evaluators at six centers were trained and certified to perform the Bayley-III. Here, we report 6- and 12-month follow-up of two subsets of these boys.Nineteen boys (1.9 ± 0.8 years) were assessed at baseline and 6 months. Twelve boys (1.5 ± 0.8 years) were assessed at baseline, 6, and 12 months.Gross motor scores were lower at baseline compared with published controls (6.2 ± 1.7; normal 10 ± 3; P < 0.0001) and revealed a further declining trend to 5.7 ± 1.7 (P = 0.20) at 6 months. Repeated measures analysis of the 12 boys monitored for 12 months revealed that gross motor scores, again low at baseline (6.6 ± 1.7; P < 0.0001), declined at 6 months (5.9 ± 1.8) and further at 12 months (5.3 ± 2.0) (P = 0.11). Cognitive and language scores were lower at baseline compared with normal children (range, P = 0.002-<0.0001) and did not change significantly at 6 or 12 months (range, P = 0.89-0.09). Fine motor skills, also low at baseline, improved >1 year (P = 0.05).Development can reliably be measured in infants and young boys with Duchenne muscular dystrophy across time using the Bayley-III. Power calculations using these data reveal that motor development may be used as an outcome measure.

Abstract

Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D.

Abstract

The onset and symptoms of the myotonic dystrophies are diverse, complicating their diagnoses and limiting a comprehensive approach to their clinical care. This report analyzes the diagnostic delay (time from onset of first symptom to diagnosis) in a large sample of myotonic dystrophy (DM) patients enrolled in the US National Registry [679 DM type 1 (DM1) and 135 DM type 2 (DM2) patients]. Age of onset averaged 34.0 ± 14.1 years in DM2 patients compared to 26.1 ± 13.2 years in DM1 (p

Abstract

Duchenne muscular dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTR(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress. Treatment with antioxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTR(KO) mice. In corroboration, all four of the DMD patients analysed had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.

Abstract

Therapeutic trials in Duchenne Muscular Dystrophy (DMD) exclude young boys because traditional outcome measures rely on cooperation. The Bayley III Scales of Infant and Toddler Development (Bayley III) have been validated in developing children and those with developmental disorders but have not been studied in DMD. Expanded Hammersmith Functional Motor Scale (HFMSE) and North Star Ambulatory Assessment (NSAA) may also be useful in this young DMD population. Clinical evaluators from the MDA-DMD Clinical Research Network were trained in these assessment tools. Infants and boys with DMD (n=24; 1.9±0.7years) were assessed. The mean Bayley III motor composite score was low (82.8±8; p⩽.0001) (normal=100±15). Mean gross motor and fine motor function scaled scores were low (both p⩽.0001). The mean cognitive comprehensive (p=.0002), receptive language (p⩽.0001), and expressive language (p=.0001) were also low compared to normal children. Age was negatively associated with Bayley III gross motor (r=-0.44; p=.02) but not with fine motor, cognitive, or language scores. HFMSE (n=23) showed a mean score of 31±13. NSAA (n=18 boys; 2.2±0.4years) showed a mean score of 12±5. Outcome assessments of young boys with DMD are feasible and in this multicenter study were best demonstrated using the Bayley III.

Abstract

OBJECTIVE: Duchenne muscular dystrophy (DMD) displays a clinical range that is not fully explained by the primary DMD mutations. Ltbp4, encoding latent transforming growth factor-β binding protein 4, was previously discovered in a genome-wide scan as a modifier of murine muscular dystrophy. We sought to determine whether LTBP4 genotype influenced DMD severity in a large patient cohort. METHODS: We analyzed nonsynonymous single nucleotide polymorphisms (SNPs) from human LTBP4 in 254 nonambulatory subjects with known DMD mutations. These SNPs, V194I, T787A, T820A, and T1140M, form the VTTT and IAAM LTBP4 haplotypes. RESULTS: Individuals homozygous for the IAAM LTBP4 haplotype remained ambulatory significantly longer than those heterozygous or homozygous for the VTTT haplotype. Glucocorticoid-treated patients who were IAAM homozygotes lost ambulation at 12.5 ± 3.3 years compared to 10.7 ± 2.1 years for treated VTTT heterozygotes or homozygotes. IAAM fibroblasts exposed to transforming growth factor (TGF) β displayed reduced phospho-SMAD signaling compared to VTTT fibroblasts, consistent with LTBP4' role as a regulator of TGFβ. INTERPRETATION: LTBP4 haplotype influences age at loss of ambulation, and should be considered in the management of DMD patients. ANN NEUROL 2013.

Abstract

Diffusion tensor imaging was used to evaluate cerebral white matter in 16 patients (ages 9-18) with myotonic dystrophy type 1 compared to 15 matched controls. Patients with myotonic dystrophy showed abnormalities in mean diffusivity compared to controls in frontal, temporal, parietal, and occipital white matter and in all individual tracts examined. Whole cerebrum mean diffusivity was 8.6 % higher overall in patients with myotonic dystrophy compared to controls. Whole cerebrum fractional anisotropy was also abnormal (10.8 % low overall) in all regions and tracts except corticospinal tracts. Follow-up analysis of parallel and perpendicular diffusivity suggests possible relative preservation of myelin in corticospinal tracts. Correlations between Wechsler working memory performance and mean diffusivity were strong for all regions. Frontal and temporal fractional anisotropy were correlated with working memory as well. Results are consistent with earlier studies demonstrating that significant white matter disturbances are characteristic in young patients with myotonic dystrophy and that these abnormalities are associated with the degree of working memory impairment seen in this disease.

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease with an unclear genetic mechanism. Most patients have a contraction of the D4Z4 macrosatellite repeat array at 4qter, which is thought to cause partial demethylation (FSHD1) of the contracted allele. Demethylation has been surveyed at 3 restriction enzyme sites in the first repeat and only a single site across the entire array, and current models postulate that a generalized D4Z4 chromatin alteration causes FSHD. The background of normal alleles has confounded the study of epigenetic alterations; however, rare patients (FSHD2) have a form of the disease in which demethylation is global, i.e., on all D4Z4 elements throughout the genome. Our objective was to take advantage of the global nature of FSHD2 to identify where disease-relevant methylation changes occur within D4Z4.Using bisulfite sequencing of DNA from blood and myoblast cells, methylation levels at 74 CpG sites across 3 disparate regions within D4Z4 were measured in FSHD2 patients and controls.We found that rates of demethylation caused by FSHD2 are not consistent across D4Z4. We identified a focal region of extreme demethylation within a 5' domain, which we named DR1. Other D4Z4 regions, including the DUX4 ORF, were hypomethylated but to a much lesser extent.These data challenge the simple view that FSHD is caused by a broad "opening" of D4Z4 and lead us to postulate that the region of focal demethylation is the site of action of the key D4Z4 chromatin regulatory factors that go awry in FSHD.

Abstract

This study sought to compare the effectiveness and safety of an angiotensin converting enzyme inhibitor (ACE-I) (lisinopril) vs. an angiotensin receptor blocker (ARB) (losartan) for the treatment of cardiomyopathy (CM) in boys with Duchenne muscular dystrophy (DMD).Development of CM is universal in boys with DMD. ACE-I and ARB have both been suggested as effective treatment options. ARBs have been associated with skeletal muscle regeneration in a mouse model of DMD. The question of which, if either, is more effective for CM treatment in DMD remains. The purpose of this multicenter double-blind prospective study was to compare efficacy and safety of lisinopril versus losartan in the treatment of newly diagnosed CM in boys with DMD.Echocardiographic technician inter- and intraobserver variability were tested on 2 separate days on 2 different boys with DMD CM. Results were compared with paired t-testing. Twenty-two boys with newly diagnosed DMD CM (echocardiographic ejection fraction (EF) 10% EF drop. Three boys in the aCE-I group had 3 visits, due to study funding termination. Two were withdrawn because of low EF. All their data are included in the analysis for as long as they remained in the study. Mean EF's were similar at baseline (47.5%- ACE-I, 48.4%- ARB). After 1 year each group significantly improved to 54.6% and 55.2% respectively (p=.02). There was no difference between the 2 treatment groups at 1 year.Inter-observer and intra-observer reliability studies showed no differences between echocardiographers on serial examinations. EF improved equally in the two groups. There is no therapeutic difference in EF improvement between lisinopril and losartan over the one-year duration for treatment of boys with DMD-related CM.ClinicalTrials.gov NCT01982695.

Abstract

Pathophysiological mechanisms underlying the clinically devastating CNS features of myotonic dystrophy (DM) remain more enigmatic and controversial than do the muscle abnormalities of this common form of muscular dystrophy. To better define CNS and cranial muscle changes in DM, we used quantitative volumetric and diffusion tensor MRI methods to measure cerebral and masticatory muscle differences between controls (n=5) and adults with either congenital (n=5) or adult onset (n=5) myotonic dystrophy type 1 and myotonic dystrophy type 2 (n=5). Muscle volumes were diminished in DM1 and strongly correlated with reduced white matter integrity and gray matter volume. Moreover, correlation of reduced fractional anisotropy (white matter integrity) and gray matter volume in both DM1 and DM2 suggests that these abnormalities may share a common underlying pathophysiological mechanism. Further quantitative temporal and spatial characterization of these features will help delineate developmental and progressive neurological components of DM, and help determine the causative molecular and cellular mechanisms.

Abstract

In 1994, Ranum and colleagues identified a ten-generation American kindred with a relatively mild autosomal dominant form of spinocerebellar ataxia (Ranum et al., 1994). The mutation was mapped to the centromeric region of chromosome 11, and the disorder designated SCA5 (Ranum et al., 1994). Using a multifaceted mapping approach, Ikeda et al. (2006) discovered that β-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in the American kindred and two additional independently reported SCA5 families. The American and French families have separate in-frame deletions of 39 and 15 bp, respectively, in the third of 17 spectrin repeat motifs. A third mutation, found in a German family, is located in the second calponin homology domain, a region known to bind actin and Arp1. Consistent with Purkinje cell degeneration in SCA5, β-III spectrin is highly expressed in cerebellar Purkinje cells. TIRF microscopy performed on cell lines transiently transfected with mutant or wild-type spectrin shows that mutant β-III spectrin fails to stabilize the glutamate transporter EAAT4 at the plasma membrane. Additionally, marked differences in EAAT4 and GluRδ2 were found by protein blot and cell fractionation in SCA5 autopsy tissue. This review summarizes data showing that β-III spectrin mutations are a novel cause of neurodegenerative disease, which may affect the stabilization or trafficking of membrane proteins.

Abstract

To perform a double-blind, randomized study comparing efficacy and safety of daily and weekend prednisone in boys with Duchenne muscular dystrophy (DMD).A total of 64 boys with DMD who were between 4 and 10 years of age were randomized at 1 of 12 centers of the Cooperative International Neuromuscular Research Group. Efficacy and safety of 2 prednisone schedules (daily 0.75 mg/kg/day and weekend 10 mg/kg/wk) were evaluated over 12 months.Equivalence was met for weekend and daily dosing of prednisone for the primary outcomes of quantitative muscle testing (QMT) arm score and QMT leg score. Secondary strength scores for QMT elbow flexors also showed equivalence between the 2 treatment groups. Overall side effect profiles of height and weight, bone density, cataract formation, blood pressure, and behavior, analyzed at 12 months, did not differ between weekend and daily dosing of prednisone.Weekend dosing of prednisone is equally beneficial to the standard daily dosing of prednisone. Analysis of side effect profiles demonstrated overall tolerability of both dosing regimens.This study provides Class I evidence that weekend prednisone dosing is as safe and effective as daily prednisone in preserving muscle strength and preventing body mass index increases in boys with DMD over a 12-month period.

Abstract

Myotonic dystrophy is an RNA gain-of-function disease caused by expanded CUG or CCUG repeats, which sequester the RNA binding protein MBNL1. Here we describe a newly discovered function for MBNL1 as a regulator of pre-miR-1 biogenesis and find that miR-1 processing is altered in heart samples from people with myotonic dystrophy. MBNL1 binds to a UGC motif located within the loop of pre-miR-1 and competes for the binding of LIN28, which promotes pre-miR-1 uridylation by ZCCHC11 (TUT4) and blocks Dicer processing. As a consequence of miR-1 loss, expression of GJA1 (connexin 43) and CACNA1C (Cav1.2), which are targets of miR-1, is increased in both DM1- and DM2-affected hearts. CACNA1C and GJA1 encode the main calcium- and gap-junction channels in heart, respectively, and we propose that their misregulation may contribute to the cardiac dysfunctions observed in affected persons.

Abstract

Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping.

Abstract

Diffusion tensor imaging was used to evaluate cerebral white matter in eight patients (ages 10-17), with myotonic dystrophy type 1 (3 congenital-onset, 5 juvenile-onset) compared to eight controls matched for age and sex. Four regions of interest were examined: inferior frontal, superior frontal, supracallosal, and occipital. The myotonic dystrophy group showed white matter abnormalities compared to controls in all regions. All indices of white matter integrity were abnormal: fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity. With no evidence of regional variation, correlations between whole cerebrum white matter fractional anisotropy and neurocognitive functioning were examined in the patients. Strong correlations were observed between whole cerebrum fractional anisotropy and full-scale intelligence and a measure of executive functioning. Results indicate that significant white matter abnormality is characteristic of young patients with myotonic dystrophy type 1 and that the white matter abnormality seen with neuroimaging has implications for cognitive functioning.

Abstract

Obesity is a major public health concern in children. Obesity occurs frequently in boys with Duchenne muscular dystrophy (DMD), complicating treatment and impairing functioning. Parent-focused interventions to facilitate weight loss have been successful in other pediatric samples but have not been studied with this population. The current investigation examined the feasibility and potential efficacy of parent-focused treatment to improve healthy eating and physical activity of parents and eating and weight in their sons with DMD. Three families participated in this case series. Resulting changes in body weight among boys with DMD were an outcome variable. Findings indicate inconsistent changes in boys' weight, decreases in parent weight, increases in healthy foods available in the home, and increases in children's perceived quality of life. Participant ratings of treatment suitability and satisfaction were generally favorable. These preliminary findings support the use of parent-focused psychoeducation for the treatment of obesity in children with DMD.

Abstract

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with "private" mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55.

Abstract

The identification of genes for monogenic disorders has proven to be highly effective for understanding disease mechanisms, pathways and gene function in humans. Nevertheless, while thousands of Mendelian disorders have not yet been mapped there has been a trend away from studying single-gene disorders. In part, this is due to the fact that many of the remaining single-gene families are not large enough to map the disease locus to a single site in the genome. New tools and approaches are needed to allow researchers to effectively tap into this genetic gold-mine. Towards this goal, we have used haploid cell lines to experimentally validate the use of high-density single nucleotide polymorphism (SNP) arrays to define genome-wide haplotypes and candidate regions, using a small amyotrophic lateral sclerosis (ALS) family as a prototype. Specifically, we used haploid-cell lines to determine if high-density SNP arrays accurately predict haplotypes across entire chromosomes and show that haplotype information significantly enhances the genetic information in small families. Panels of haploid-cell lines were generated and a 5 centimorgan (cM) short tandem repeat polymorphism (STRP) genome scan was performed. Experimentally derived haplotypes for entire chromosomes were used to directly identify regions of the genome identical-by-descent in 5 affected individuals. Comparisons between experimentally determined and in silico haplotypes predicted from SNP arrays demonstrate that SNP analysis of diploid DNA accurately predicted chromosomal haplotypes. These methods precisely identified 12 candidate intervals, which are shared by all 5 affected individuals. Our study illustrates how genetic information can be maximized using readily available tools as a first step in mapping single-gene disorders in small families.

Abstract

Myotonic dystrophy type 2 (DM2) is caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the ZNF9 gene on chromosome 3q21. All studied DM2 mutations have been reported in Caucasians and share an identical haplotype, suggesting a common founder. We identified a Japanese patient with DM2 and showed that the affected haplotype is distinct from the previously identified DM2 haplotype shared among Caucasians. These data strongly suggest that DM2 expansion mutations originate from separate founders in Europe and Japan and are more widely distributed than previously recognized.

Abstract

The SCN8A gene on chromosome 12q13 encodes the voltage gated sodium channel Na(v)1.6, which is widely expressed in neurons of the CNS and PNS. Mutations in the mouse ortholog of SCN8A result in ataxia and other movement disorders.We screened the 26 coding exons of SCN8A in 151 patients with inherited or sporadic ataxia.A 2 bp deletion in exon 24 was identified in a 9 year old boy with mental retardation, pancerebellar atrophy, and ataxia. This mutation, Pro1719ArgfsX6, introduces a translation termination codon into the pore loop of domain 4, resulting in removal of the C-terminal cytoplasmic domain and predicted loss of channel function. Three additional heterozygotes in the family exhibit milder cognitive and behavioural deficits including attention deficit hyperactivity disorder (ADHD). No additional occurrences of this mutation were observed in 625 unrelated DNA samples (1250 chromosomes).The phenotypes of the heterozygous individuals suggest that mutations in SCN8A may result in motor and cognitive deficits of variable expressivity, but the study was limited by lack of segregation in the small pedigree and incomplete information about family members. Identification of additional families will be required to confirm the contribution of the SCN8A mutation to the clinical features in ataxia, cognition and behaviour disorders.

Abstract

Myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion mutation in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mean expansion size in patients is larger than for DM1 or any previously reported disorder (mean=5000 CCTGs; range=75-11 000), and similar to DM1, repeats containing ribonuclear inclusions accumulate in affected DM2 tissue. Although an RNA gain-of-function mechanism involving DM1 CUG or DM2 CCUG expansion transcripts is now well established, still debated are the potential role that flanking sequences within the DMPK 3'-UTR may have on disease pathogenesis and whether or not decreased expression of DMPK, ZNF9 or neighboring genes at these loci contribute to disease. To address these questions in DM2, we have examined the nucleic acid content of the ribonuclear inclusions and the effects of these large expansions on ZNF9 expression. Using cell lines either haploid or homozygous for the expansion, as well as skeletal muscle biopsy tissue, we demonstrate that pre-mRNAs containing large CCUG expansions are normally spliced and exported from the nucleus, that the expansions do not decrease ZNF9 expression at the mRNA or protein level, and that the ribonuclear inclusions are enriched for the CCUG expansion, but not intronic flanking sequences. These data suggest that the downstream molecular effects of the DM2 mutation are triggered by the accumulation of CCUG repeat tract alone.

Abstract

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Abstract

The general model that dominant diseases are caused by mutations that result in a gain or change in function of the corresponding protein was challenged by the discovery that the myotonic dystrophy type 1 mutation is a CTG expansion located in the 3' untranslated portion of a kinase gene. The subsequent discovery that a similar transcribed but untranslated CCTG expansion in an intron causes the same multisystemic features in myotonic dystrophy type 2 (DM2), along with other developments in the DM1 field, demonstrate a mechanism in which these expansion mutations cause disease through a gain of function mechanism triggered by the accumulation of transcripts containing CUG or CCUG repeat expansions. A similar RNA gain of function mechanism has also been implicated in fragile X tremor ataxia syndrome (FXTAS) and may play a role in pathogenesis of other non-coding repeat expansion diseases, including spinocerebellar ataxia type 8 (SCA8), SCA10, SCA12 and Huntington disease-like 2.

Abstract

Pathogenic repeat expansions were initially identified as causing either a loss of gene product, such as in fragile X mental retardation, or an expansion of a polyglutamine region of a protein, as was first shown in spinobulbar muscular atrophy (Kennedy's disease). The pathogenic effect of the repeat expansion in myotonic dystrophy type 1, however, has been controversial because it does not encode a protein but nonetheless results in a highly penetrant dominant disease. Clinical and molecular characterization of myotonic dystrophy types 1 and 2 have now demonstrated a novel disease mechanism involving pathogenic effects of repeat expansions that are expressed in RNA but are not translated into protein.

Abstract

Myotonic dystrophy (dystrophia myotonica, DM) is the most common form of muscular dystrophy in adults. The presence of two genetic forms of this complex multisystemic disease (DM1 and DM2) was unrecognized until the genetic cause of DM1 was identified in 1992. The fact that the DM1 mutation is an untranslated CTG expansion led to extended controversy about the molecular pathophysiology of this disease. When the DM2 mutation was identified in 2001 as being a similarly untranslated CCTG expansion, the molecular and clinical parallels between DM1 and DM2 substantiated the role of a novel mechanism in generating the unusual constellation of clinical features seen in these diseases: the repeat expansions expressed at the RNA level alter RNA processing, at least in part by interfering with alternative splicing of other genes. For example, in both DM1 and DM2, altered splicing of chloride channel and insulin receptor transcripts leads to myotonia and insulin resistance, respectively. Although other mechanisms may underlie the differences between DM1 and DM2, the pathogenic effects of the RNA mechanism are now clear, which will facilitate development of appropriate treatments.

Abstract

Medical records and follow-up data were reviewed in 297 genetically proven myotonic dystrophy type 2 (DM2) patients. Patients were selected by the criteria of cardiac sudden death before age 45. Sudden death occurred in four patients, three of whom were cardiological asymptomatic, and one with a history of heart failure. Cardiac histopathology showed dilated cardiomyopathy in all, and conduction system fibrosis in two patients. Pathogenetic CCUG ribonuclear inclusions were demonstrable in cardiomyocytes.

Abstract

We reported elsewhere that an untranslated CTG expansion causes the dominantly inherited neurodegenerative disorder spinocerebellar ataxia type 8 (SCA8). SCA8 shows a complex inheritance pattern with extremes of incomplete penetrance, in which often only one or two affected individuals are found in a given family. SCA8 expansions have also been found in control chromosomes, indicating that separate genetic or environmental factors increase disease penetrance among SCA8-expansion-carrying patients with ataxia. We describe the molecular genetic features and disease penetrance of 37 different families with SCA8 ataxia from the United States, Canada, Japan, and Mexico. Haplotype analysis using 17 STR markers spanning an approximately 1-Mb region was performed on the families with ataxia, on a group of expansion carriers in the general population, and on psychiatric patients, to clarify the genetic basis of the reduced penetrance and to investigate whether CTG expansions among different populations share a common ancestral background. Two major ancestrally related haplotypes (A and A') were found among white families with ataxia, normal controls, and patients with major psychosis, indicating a common ancestral origin of both pathogenic and nonpathogenic SCA8 expansions among whites. Two additional and distinct haplotypes were found among a group of Japanese families with ataxia (haplotype B) and a Mexican family with ataxia (haplotype C). Our finding that SCA8 expansions on three independently arising haplotypes are found among patients with ataxia and cosegregate with ataxia when multiple family members are affected further supports the direct role of the CTG expansion in disease pathogenesis.

Abstract

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.

Abstract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.

Abstract

Myotonic dystrophy types 1 (DM1) and 2 (DM2/proximal myotonic myopathy PROMM) are dominantly inherited disorders with unusual multisystemic clinical features. The authors have characterized the clinical and molecular features of DM2/PROMM, which is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene.Three-hundred and seventy-nine individuals from 133 DM2/PROMM families were evaluated genetically, and in 234 individuals clinical and molecular features were compared.Among affected individuals 90% had electrical myotonia, 82% weakness, 61% cataracts, 23% diabetes, and 19% cardiac involvement. Because of the repeat tract's unprecedented size (mean approximately 5,000 CCTGs) and somatic instability, expansions were detectable by Southern analysis in only 80% of known carriers. The authors developed a repeat assay that increased the molecular detection rate to 99%. Only 30% of the positive samples had single sizeable expansions by Southern analysis, and 70% showed multiple bands or smears. Among the 101 individuals with single expansions, repeat size did not correlate with age at disease onset. Affected offspring had markedly shorter expansions than their affected parents, with a mean size difference of -17 kb (-4,250 CCTGs).DM2 is present in a large number of families of northern European ancestry. Clinically, DM2 resembles adult-onset DM1, with myotonia, muscular dystrophy, cataracts, diabetes, testicular failure, hypogammaglobulinemia, and cardiac conduction defects. An important distinction is the lack of a congenital form of DM2. The clinical and molecular parallels between DM1 and DM2 indicate that the multisystemic features common to both diseases are caused by CUG or CCUG expansions expressed at the RNA level.

Abstract

We previously reported that a transcribed but untranslated CTG expansion causes a novel form of ataxia, spinocerebellar ataxia type 8 (SCA8) (Koob et al., 1999). SCA8 was the first example of a dominant spinocerebellar ataxia that is not caused by the expansion of a CAG repeat translated into a polyglutamine tract. This slowly progressive form of ataxia is characterized by dramatic repeat instability and a high degree of reduced penetrance. The clinical and genetic features of the disease are discussed below.

Abstract

We initiated a randomized, double-blinded, placebo-controlled trial of intravenous immunoglobulin (IVIG) treatment in myasthenia gravis (MG). Patients received IVIG 2 gm/kg at induction and 1 gm/kg after 3 weeks vs. 5% albumin placebo. The primary efficacy measurement was the change in the quantitative MG Score (QMG) at day 42. Fifteen patients were enrolled (6 to IVIG; 9 to placebo) before the study was terminated because of insufficient IVIG inventories. At day 42, there was no significant difference in primary or secondary outcome measurements between the two groups. In a subsequent 6-week open-label study of IVIG, positive trends were observed.

Abstract

Myotonic dystrophy (DM) is a dominantly inherited disorder with a peculiar pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1 on chromosome 19, and DM2 on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophica myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes DM1 has been a matter of controversy. The recent discovery that DM2 is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain of function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes.

Abstract

To obtain an objective measure of muscle force in periodic paralysis, we studied ankle dorsiflexion torque during induced paralytic attacks in hyperkalemic and hypokalemic patients. SUBJECTS, PATIENTS, AND METHODS: Dorsiflexor torque after peroneal nerve stimulation was recorded during provocative tests on 5 patients with hypokalemic or hyperkalemic disorders and on 2 control subjects (1995-2001). Manual strength assessment was simultaneously performed in a blinded fashion. Standardized provocation procedures were used.The loss of torque in hyperkalemic patients roughly paralleled the loss of clinically detectable strength, whereas in the hypokalemic patients, pronounced torque loss occurred well before observed clinical effects. No dramatic changes occurred in the control subjects. Torque amplitude decreased more than 70% in all patients during the provocation tests; such decreases were associated with alterations induced in serum potassium concentrations.Stimulated torque measurement offers several advantages in characterizing muscle dysfunction in periodic paralysis: (1) it is independent of patient effort; (2) it can show a definitely abnormal response early during provocative maneuvers; and (3) characteristics of muscle contraction can be measured that are unobservable during voluntary contraction. Stimulated torque measurements can characterize phenotypic muscle function in neuromuscular diseases.

Abstract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19q13 (DM1) or 3q21 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). Several mechanisms have been invoked to explain how this mutation, which does not alter the protein-coding portion of a gene, causes the specific constellation of clinical features characteristic of DM. We now report that DM2 is caused by a CCTG expansion (mean approximately 5000 repeats) located in intron 1 of the zinc finger protein 9 (ZNF9) gene. Parallels between these mutations indicate that microsatellite expansions in RNA can be pathogenic and cause the multisystemic features of DM1 and DM2.

Abstract

Lipid-enveloped viruses such as HIV, HBV, and HCV can be inactivated by treatment with solvents and detergents. HAV and human parvovirus B19 lack lipid envelopes and are not inactivated. Solvent/detergent-treated pooled plasma (S/D plasma) contains neutralizing antibodies, but it is not known whether the parvovirus B19 antibody content is sufficient to prevent transmission of the disease. A patient is described who developed a clinical illness due to parvovirus B19 infection after the infusion of S/D plasma.A 36-year-old woman with myasthenia gravis underwent five plasma exchange procedures from January 15 to January 25, 1999, using albumin, except for 5 units of SD plasma given because of a low fibrinogen level. Four of the 5 units were implicated in a recall after high levels of parvovirus B19 DNA were found in several lots. Two weeks after the infusion, the patient developed fatigue, a rash, and severe polyarthralgias. Parvovirus B19 IgG and IgM antibody titers were consistent with an acute infection.Clinically apparent parvovirus B19 infection can follow the use of S/D plasma that contains high levels of parvovirus B19 DNA.

Abstract

To compare the clinical and genetic features of the seven-generation family (MN-A) used to define the spinocerebellar ataxia 8 (SCA8) locus.The authors recently described an untranslated CTG expansion that causes a novel form of SCA (SCA8) characterized by reduced penetrance and complex patterns of repeat instability.Clinical and molecular features of 82 members of the MN-A family were evaluated by neurologic examination, quantitative dexterity testing, and, in some individuals, MRI and sperm analyses.SCA8 is a slowly progressive, predominantly cerebellar ataxia with marked cerebellar atrophy, affecting gait, swallowing, speech, and limb and eye movements. CTG tracts are longer in affected (mean = 116 CTG repeats) than in unaffected expansion carriers (mean = 90, p < 10-8). Quantitative dexterity testing did not detect even subtle signs of ataxia in unaffected expansion carriers. Surprisingly, all 21 affected MN-A family members inherited an expansion from their mothers. The maternal penetrance bias is consistent with maternal repeat expansions yielding alleles above the pathogenic threshold in the family (>107 CTG) and paternal contractions resulting in shorter alleles. Consistent with the reduced penetrance of paternal transmissions, CTG tracts in all or nearly all sperm (84 to 99) are significantly shorter than in the blood (116) of an affected man.The biologic relationship between repeat length and ataxia indicates that the CTG repeat is directly involved in SCA8 pathogenesis. Diagnostic testing and genetic counseling are complicated by the reduced penetrance, which often makes the inheritance appear recessive or sporadic, and by interfamilial differences in the length of a stable (CTA)n tract preceding the CTG repeat.

Abstract

We recently described an untranslated CTG expansion that causes a previously undescribed form of spinocerebellar ataxia (SCA8). The SCA8 CTG repeat is preceded by a polymorphic but stable CTA tract, with the configuration (CTA)(1-21)(CTG)(n). The CTG portion of the repeat is elongated on pathogenic alleles, which nearly always change in size when transmitted from generation to generation. To better understand the reduced penetrance and maternal penetrance bias associated with SCA8 we analyzed the sequence configurations and instability patterns of the CTG repeat in affected and unaffected family members. In contrast to other triplet repeat diseases, expanded alleles found in affected SCA8 individuals can have either a pure uninterrupted CTG repeat tract or an allele with one or more CCG, CTA, CTC, CCA or CTT interruptions. Surprisingly, we found six different sequence configurations of the CTG repeat on expanded alleles in a seven generation family. In two instances duplication of CCG interruptions occurred over a single generation and in other instances duplications that had occurred in different branches of the family could be inferred. We also evaluated SCA8 instability in sperm samples from individuals with expansions ranging in size from 80 to 800 repeats in blood. Surprisingly the SCA8 repeat tract in sperm underwent contractions, with nearly all of the resulting expanded alleles having repeat lengths of <100 CTGs, a size that is not often associated with disease. These en masse repeat contractions in sperm likely underlie the reduced penetrance associated with paternal transmission.

Abstract

We report the clinical and genetic characteristics of a five-generation family (MN1) with an unusual form of myotonic dystrophy (DM). Affected individuals have clinical features that are similar to DM including myotonia, distal weakness, frontal balding, polychromatic cataracts, infertility and cardiac arrhythmias. Genetic analyses reveal that affected individuals do not have the CTG expansion associated with DM, nor is the disease locus linked to the DM region of chromosome 19. We have also excluded the MN1 disease locus from the chromosomal regions containing the genes for the muscle sodium (alpha- and beta-subunits) and chloride channels, both of which are involved in other myotonic disorders. We have recently mapped the disease locus (DM2) in this family to a 10 cM region of chromosome 3q [Ranum LPW, Rasmussen PF, Benzow KA, Koob MD, Day JW. Nat Genet 1998;19:196-198]. The genetically distinct form of myotonic dystrophy in the MN1 kindred shares some of the clinical features of previously reported families with proximal myotonic myopathy (PROMM). The size of the MN1 family (25 affected individuals) makes it a unique resource for both clinical and genetic studies. This second form of myotonic dystrophy may help resolve the confusion that remains about how the CTG repeat expansion in the 3' untranslated portion of the myotonin protein kinase gene causes the multisystem involvement of DM.

Abstract

We report the mapping of a second myotonic dystrophy locus, myotonic dystrophy type 2 (DM2). Myotonic dystrophy (DM) is a multi-system disease and the most common form of muscular dystrophy in adults. In 1992, DM was shown to be caused by an expanded CTG repeat in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) on chromosome 19 (refs 2-6). Although several theories have been put forth to explain how the CTG expansion causes the broad spectrum of clinical features associated with DM, it is not understood how this mutation, which does not alter the protein-coding region of a gene, causes an affect at the cellular level. We have identified a five-generation family (MN1) with a genetically distinct form of myotonic dystrophy. Affected members exhibit remarkable clinical similarity to DM (myotonia, proximal and distal limb weakness, frontal balding, cataracts and cardiac arrhythmias) but do not have the chromosome-19 D CTG expansion. We have mapped the disease locus (DM2) of the MN1 family to a 10-cM region of chromosome 3q. Understanding the common molecular features of two different forms of the disease should shed light on the mechanisms responsible for the broad constellation of seemingly unrelated clinical features present in both diseases.

Abstract

Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.

Abstract

While the slow onset of desensitization of nicotinic acetylcholine receptors (AChRs), relative to the rate of acetylcholine removal, excludes this kinetic state from shaping synaptic responses in normal neuromuscular transmission, its role in neuromuscular disorders has not been examined. The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder caused by point mutations in the AChR subunit-encoding genes leading to kinetically abnormal (slow) channels, reduced miniature endplate current amplitudes (MEPCs), and degeneration of the postsynaptic membrane. Because of this complicated picture of kinetic and structural change in the neuromuscular junction, it is difficult to assess the importance of the multiple factors that may be responsible for the reduced endplate current amplitudes, and ultimately the clinical syndrome. In order to address this we have used a transgenic mouse model for the SCCMS that has slow AChR ion channels and reduced endplate responsiveness in the absence of any of the degenerative changes. We found that the reduction in MEPC amplitudes in these mice could not be explained by either reduced AChR number or by reduced AChR channel conductance. Rather, we found that the mutant AChRs in situ manifested an activity-dependent reduction in sensitivity that caused diminished MEPC and endplate current amplitude with nerve stimulation. This observation demonstrates that the basis for the reduction in MEPC amplitudes in the SCCMS may be multifactorial. Moreover, these findings demonstrate that, under conditions that alter their rate of desensitization, the kinetic properties of nicotinic AChRs can control the strength of synaptic responses.

Abstract

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.

Abstract

We describe here the reliability and validity of methods to quantify involuntary muscle torque induced by non-invasive nerve stimulation. A rigid apparatus was used to hold the subject's limb in a predetermined position and confine movement to a specific direction (i.e. ankle dorsiflexion or thumb adduction). An incorporated strain gauge was used to measure isometric torque, and all data were recorded by a data acquisition program. The innervating nerves were stimulated by surface electrodes, using either single stimuli to generate a twitch, or short trains of stimuli to produce tetanic contraction of the individual muscle under study. The average peak tetanic torque generated by the dorsiflexor muscles in healthy control was 20.4 +/- 3.8 Nm and varied by 3.7% with repeated testing. The mean torque generated by the adductor pollicis muscle in controls was 1.5 +/- 0.4 Nm and varied by 4.6% with repeated testing. In patient populations significant changes in activated torque were readily quantified, and the effects of treatment can be easily assessed. Furthermore, several specific parameters of recorded isometric contractions were measured; e.g. time between stimulus and torque onset, peak rate of torque development, time to peak torque, half-relaxation time, and others (none of which are measurable when using voluntary contraction of muscle). Compared to current assessment methods, monitoring muscle torque generated by nerve stimulation improves objectivity, reliability, and quantitative capabilities. The presented method has significant potential both in diagnosing neuromuscular disorders and determining treatment efficacy.

Abstract

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a delta subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs.

Abstract

Adenosine modulation of nicotinic ACh receptor (nAChR) function was studied in primary cultures of rat skeletal muscle. Activation of the nAChR by carbachol increased extracellular adenosine concentration in a dose-dependent manner. Furthermore, carbachol activation of the nicotinic receptor resulted in a twofold increase in cAMP levels in the muscle cells. The carbachol-dependent increase in cAMP levels was inhibited by adenosine receptor antagonists as well as by nicotinic receptor antagonists. These results suggest that the increased cAMP levels were due to adenosine receptor activation by the extracellular adenosine accumulated on nAChR activation. Others have shown that desensitization of the nAChR by agonist is mediated, in part, by phosphorylation. Since we found that nicotinic cholinergic agonists also cause adenosine accumulation with concomitant cAMP increases, we determined whether the accumulated adenosine has a role in desensitization. We found that the adenosine receptor antagonist, BW1434U, significantly inhibited carbachol-induced nAChR desensitization, indicating that extracellular adenosine is involved in nAChR desensitization. Our data suggest that nAChR function is regulated via a feedback mechanism mediated by adenosine released from muscle on activation of the nAChR.

Abstract

Tetany is a disorder of increased neuronal excitability usually associated with hypocalcemia. We studied a patient with typical tetanic cramps that responded to intravenous infusion of calcium despite normal serum concentrations of total and ionized calcium. Three generations were affected by similar symptoms, suggesting that this is a dominantly inherited disorder of neuronal hyperirritability.

Abstract

Many patients with a ruptured berry aneurysm report an intense sentinel headache of sudden onset in the weeks before rupture. Such headaches have been attributed to a leak of blood, which implies that partial rupture has occurred. A case is reported of a patient who had severe headaches which seemed to be caused by an unruptured cerebral aneurysm, accompanied by diffuse cerebral vasospasm. Headache episodes with the thunderclap profile may require angiography for diagnosis even if the cerebrospinal fluid is bloodless.

Abstract

The hatchetfish Mauthner fiber is presynaptic to 8-14 large myelinated axons in the medulla; the large ('giant') synapses formed by these fibers appear to be nicotinic cholinergic. Miniature postsynaptic potentials (mPSPs) were recorded from single identified synapses. The mPSPs were averaged to more accurately determine their shape; the rise time was approximately 70 microseconds, and the fall usually was biphasic with time constants of decay for the two phases of 280 and 800 microseconds. In 25% of the records analyzed a third, slow tail of decay was seen which had an average decay constant of 4.2 ms. The biphasic decay of mPSPs largely accounts for the similar shape of the postsynaptic current following a presynaptic impulse, which is described in the accompanying paper.

Abstract

Postsynaptic currents (PSCs) at the giant synapse between Mauthner and giant fibers of the hatchetfish Gasteropelecus were studied under voltage clamp. This axo-axonic synapse lies in the central nervous system beneath the floor of the 4th ventricle where electrodes can be closely positioned both pre- and postsynaptically. Transmission is nicotonic cholinergic. The PSCs produced by Mauthner fiber impulses rise rapidly to a peak and decay in two phases; an early more rapid phase is followed by a late slower phase. The slope conductance of the peak amplitude of the PSCs declines at more inside positive potentials. The late phase of decay is exponential and voltage dependent, becoming faster for PSCs evoked at more inside positive potentials. At potentials positive to about -40 mV the late phase merges with the early phase. The decay rate constant of the slowest phase is exponentially related to voltage for potentials negative to about -10 mV, but becomes less voltage dependent for more positive potentials. The peak current is independent of whether it is evoked during inward or outward active currents of the electrically excitable membrane, and two phase decays are observed in PSCs of reduced quantal content. Thus, changes in slope conductance and two phase decays are not due to series resistance or interactions between quanta. PSCs can be modeled by a 3 state reaction scheme in which closed channels open when they bind transmitter and then can pass to a second closed state with receptor still bound such that they must return through the open state before losing their transmitter and returning to the resting, closed state.